The NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation

All of the animal experiments were conducted in accordance with the guidelines of Beijing Municipality on the Review of Welfare and Ethics of Laboratory Animals approved by the Beijing Municipality Administration Office of Laboratory Animals (BAOLA).

Experiments were conducted on murine primary microglia and BV2 microglial cells. The choice of this cell line is justified by the close similarities between BV-2 and primary microglia in mechanisms mediating microglial activation [21]. Primary microglial cell cultures were obtained from neonatal C57BL/6 mice (5 to 7 days old) as described previously [14]. Briefly, after sterilization, the brain was dissected, then the cerebral cortices were collected and digested with trypsin (0.25 %) for 15 minutes at 37°C. The digested tissue was repeatedly sucked into a pipette to obtain single cells. The cells were then passed through a 200 μm mesh and separated by centrifugation at 100 g for 5 minutes. The mixed glial cells were cultured for about 6 to 7 days, after which the cells were suspended by agitation for 12 hours on a rotary shaker (180 rpm) at 37°C and transferred to another flask. After incubation for 2 hours at 37°C, microglia had adhered to the flask. The purity of the microglial cells was approximately 90 %, as determined using anti-CD-11b antibodies. BV2 cells, a murine microglial cell line, and ANA1, a murine macrophage cell line, were obtained (Xiehe Medical University, Yuqin Liu, Cell Culture Center, Beijing, China), and cultured in a humidified incubator at 37°C with 5 % CO2 in DMEM and F12 medium (Hyclone, Logan, UT, USA) supplemented with 10 % heat-inactivated FBS (Gibco, Grand Island, NY, USA), 100 μg/ml streptomycin, 100 U/ml penicillin (Gibco), and 2 mmol/l glutamine.

PrP peptides PrP106-126 and scrambled PrP106-126 (Scr-PrP) (sequences KTNMKHMAGAAAAGAVVGGLG and AVGMHAGKGLANTAKAGAMVG, respectively), were synthesized (Sangon Bio-Tech, China). The purity of prion peptides was >95 % according to the data from the synthesizer. The peptides were dissolved in 0.1 mol/l PBS to a concentration of 1 mmol/l, and left to aggregate at 37°C for 12 hours. Experiments were conducted with final peptide concentrations of 100 μmol/l.

Microglia were primed with 300 ng/ml LPS for 3 hours, after which the culture medium was washed off and the cells were treated with the aggregated peptide PrP106-126 in culture medium. Scr-PrP was used as the negative control. Three wells were used for each group of experimental conditions. In experiments involving co-treatments with PrP106-126 plus high potassium (130 mmol/l) or NAC (15 mmol/l), cells were primed with 300 ng/ml LPS for 3 hours, and were then exposed to the indicated concentrations of the inhibitors.

Small interfering (si)RNA used for NALP3 and ASC silencing, and the scramble siRNA sequence used as control (all Qiagen, Valencia, CA, USA) were used. For siRNA transfection, cells were plated at 0.8 × 10⁵ cells/well in a 12-well plate, and transfected the next day in accordance with the manufacturer’s instructions. Briefly, on the day of transfection, 75 ng siRNA (siRNA-NALP3, siRNA-ASC and control, respectively; Qiagen) were diluted in 100 μl culture medium without serum. A volume of 3 μl of transfection reagent (HiPerfect Transfection Reagent; Qiagen) was added to the diluted siRNA and mixed by vortex. The samples were then incubated for 5 to 10 minutes at room temperature to allow the formation of transfection complexes before adding the complexes onto the cells. The decrease in NALP3 and ASC expression after treatment was checked by quantitative PCR and western blot analysis.

Cell-culture supernatants were assayed for IL-1β by ELISA using a commercial kit (Wuhan Boster Biotech) in accordance with the manufacturer’s instructions.

Total RNA was extracted from cells using the SV Total RNA Isolation System (Promega, Madison, WI, USA), and reverse transcribed into complementary (c)DNA using a commercial kit (cDNA Synthesis Kit; Fermentas, Glen Burnie, MD, USA) using oligo (dT) 18 primers in accordance with the manufacturer’s instructions. Quantitative (q)PCR was performed using a commercial mix (SYBR Green Master Mix; Bio-Rad) and a thermal cycler (DNA Engine Opticon™ 2 system; MJ Research, Waltham, MA, USA) with the primers shown in Table 1. The amplification efficiency of these primers had been established by means of calibration curves. The total volume for qPCR was 20 μl, comprised of includes 8 μl water, 0.5 μl of each primer (10 μmol/l), 10 μl Master Mix and 30 ng of cDNA. The PCR amplification was as follows: after denaturation at 94°C for 2 minutes, 40 PCR cycles of 94°C for 20 seconds, 55°C for 20 seconds, 72°C for 20 seconds, followed by 1 cycle at 84°C for 1 second appended for a single fluorescence measurement above the melting temperature of possible primer-dimers. Finally, a melting step was performed consisting of 10 seconds at 70°C and slow heating at a rate of 0.1°C per second up to 95°C with continuous fluorescence measurement. Quantification was performed using the comparative C_T method (2^-ΔΔCT) [22]. All samples were analyzed in triplicate.

After treatment of microglia cells, the culture medium was discarded, and the cytoplasmic and nuclei proteins were extracted using a protein extract kit (Cytoplasmic and Nuclear Protein Extraction Kit; Wuhan Boster Biotech). Equal amounts of protein (40 μg in each lane) were separated by SDS-PAGE on 12 % gels, and the separated proteins were transferred onto a nitrocellulose membrane. Nonspecific binding sites were blocked by incubating the membrane with 5 % fat-free dried milk in Tris-buffered saline (TBS-T: 10 mmol/l Tris, 0.15 mol/l NaCL, 0.05 % Tween-20, pH of the solution adjusted to 7.5). Rabbit anti-NF-κB p65 (1:500), anti-caspase-1 (1:500), anti-NALP3 (1:5000), or anti-ASC (1:500) antibodies were added and incubated at 4°C overnight. Membranes were washed with TBS-T, and then incubated with the secondary antibody, either goat anti-mouse IgG or anti-rabbit IgG horseradish peroxidase-conjugated antibody (1:5000). Bands of immunoreactive protein were visualized after membrane incubation with enhanced chemifluorescence (ECF) reagent for 5 minutes, on an image system (Versadoc; Bio-Rad). The blot was stripped and reprobed with anti-β-actin (for cytoplasmic extracts) or anti-Max (for nuclear extracts) to estimate the total amount of protein loaded in gel.

All assays were performed on three separate occasions. Data are expressed as means ± S.D. All comparisons for parametric data were made using Student’s t test or one-way ANOVA followed by post hoc Turkey’s test, Nonparametric data (ELISA of the primary microglia) were analyzed by the Kruskal–Wallis ANOVA test followed by the Nemenyi test for post hoc analyses. SPSS software (version 13.0: SPSS Inc., Chicago, IL, USA) was used, and P < 0.05 was considered significant.

To investigate the mechanisms of PrP106-126 induced release of IL-1β, we incubated primary mouse microglial cells and BV2 cell lines with PrP106-126 and its scrambled form, Scr-PrP. Because pro-IL-1β is not constitutively expressed in microglia [23], the cells were primed with 300 ng/ml LPS for 3 hours to induce pro-IL-1β synthesis and to mimic the chronic activation of microglia in prion disease. Treatment of LPS-primed BV2 microglia and primary microglia with PrP106-126 led to a significant increase in IL-1β release. At all time points examined, the IL-1β level was significantly higher in cells treated with PrP106-126 than in those treated with Scr-PrP or PBS (Figure 1).

Similarly, only the PrP106-126 treatment led to caspase-1 activation in LPS-primed BV2 microglia and the murine macrophage cell line, ANA1, as indicated by the cleavage of caspase-1 to its active p20 subunit. No caspase-1 cleavage was seen in LPS-primed BV2 microglia or in ANA1 treated with PBS or Scr-PrP (Figure 2). These results indicate that PrP106-126 induces IL-1β release and activates caspase-1 from microglia.

To investigate the involvement of NALP3 inflammasome activation in PrP106-126-induced microglial activation, we first examined the effect of PrP106-126 treatment on the mRNA expression of NALP3 and ASC in BV2 microglia. PrP106-126 treatment significantly upregulated the mRNA expression of both NALP3 and ASC in microglia (Figure 3a,b). The mRNA level of NALP3 was significantly higher in PrP106-126-treated microglia than in PBS-treated microglia at all time points examined, while ASC expression was upregulated only at 24 and 36 hours after PrP106-126 stimulation. The PrP106-126-induced upregulation of NALP3 and ASC indicates an active participation of NALP3 inflammasome in PrP106-126-induced microglial activation.

To elucidate the role of the NALP3 inflammasome in PrP106-126-induced microglial activation, we examined the role of the NALP3 inflammasome in PrP106-126-induced IL-1β release. Because the NALP3 inflammasome is a multiprotein complex that consists of NALP3, ASC, and pro-caspase-1 [24, 25], we analyzed the effect of siRNA-mediated disruption of NALP3 or ASC on IL-1β release in PrP106-126-treated microglia. The efficiency of siRNA-mediated disruption was evaluated at 24 and 48 hours after siRNA transfection by qPCR (#x2009;4a) and western blot analysis (Figure 4b), respectively. Expression of NALP3 and ASC was significantly downregulated both at the mRNA (76 % and 80 %, respectively) and protein levels after siRNA transfection.

Following NALP3 or ASC disruption, BV2 cells were primed with 300 ng/ml LPS for 3 hours before PrP106-126 treatment. The cells were then exposed to PrP106-126, and the cell-culture supernatants were collected at 6 hours after PrP106-126 exposure and assayed for IL-1β by ELISA. Knock-down of either NALP3 or ASC significantly reduced the release of IL-1β after exposure to PrP106-126 (Figure 4c), suggesting a key role for the NALP3 inflammasome in PrP106-126- induced IL-1β release.

Several studies have shown that the NALP3 inflammasome assembly requires a low K + intracellular environment [26], and the activation of NALP3 inflammasome is reportedly blocked by reactive oxygen species (ROS) inhibitors through a mechanism that is not well understood [27]. To determine the role of K + and ROS in PrP106-126-induced NALP3 inflammasome activation, we evaluated the effect of increasing the level of extracellular K + and of NAC (an antioxidant known for its ability to scavenge ROS), on PrP106-126-induced secretion of IL-1β and NALP3 and ASC upregulation.

A high extracellular K + concentration significantly abrogated PrP106-126-induced release of IL-1β in LPS-primed microglia. Similarly, NAC significantly blocked IL-1β activation in LPS-primed microglia stimulated with PrP106-126 (Figure 5a).

NAC significantly abrogated PrP106-126-induced NALP3 and ASC upregulation; the mRNA levels of NALP3 and ASC significantly decreased after NAC treatment in PrP106-126-treated microglia and dropped to 68 % and 54 %, respectively, of the levels seen in microglia treated with PrP106-126 only (Figure 5b). Interestingly, higher levels of extracellular K + significantly reduced the PrP106-126-induced NALP3 upregulation (64 % of the mRNA level seen in microglia treated with PrP106-126 only) but had no effect on ASC upregulation (Figure 5b). These results suggest that a low K + intracellular environment and ROS production are also required for PrP106-126-induced NALP3 inflammasome activation, and indicate that the expression of ASC and NALP3 may be regulated through closely related but not identical regulatory pathways in PrP106-126-treated microglia.

Previous studies with brain samples from mice infected with prion agent showed upregulation of multiple cytokines and chemokines, including pro-inflammatory TNF, IL-1α, transforming growth factor β, CCL2, and CCL3 [9]. To investigate the involvement of NALP3 inflammasome activation in PrP106-126-induced upregulation of pro-inflammatory cytokines and chemotactic factors, we examined the effect of siRNA-mediated silencing of NALP3 and ASC on the mRNA expression of TNF and CCL3 in LPS-primed BV2 microglia stimulated with PrP106-126.

After siRNA-mediated disruption of NALP3 or ASC, cells were treated with PrP106-126 for 12 hours, then total RNA was extracted and used to measure the level of TNF and CCL3 encoding mRNA, using qPCR. Knock-down of either NALP3 or ASC significantly reduced PrP106-126-induced upregulation of TNF- and CCL3-encoding mRNA (Figure 6a,b). The degree of downregulation caused by NALP3 inflammasome disruption was more pronounced for TNF than for CCL3 expression. These data suggest that NALP3 inflammasome activation affects TNF and CCL3 expression in PrP106-126-stimulated BV2 cell line.

The transcription factor NF-κB has been shown to play an important role in prion-induced inflammation, and PrP106-126-induced NF-κB activation has been widely reported [13, 28, 29]. In this study we also found that PrP106-126 treatment stimulated NF-κB activation as indicated by the nuclear translocation of p65 in BV2-microglia (Figure 7a).

We then examined the relevance of NF-κB activation to NALP3 activation through the analysis of the effect of NF-κB inhibitor, Bay 11–7082, on the expression of NALP3- and ASC-encoding mRNA in PrP106-126-treated BV2 microglia. The results showed that low, moderate, and high concentrations of Bay 11–7082 significantly reduced the PrP106-126-induced upregulation of NALP3- and ASC-encoding mRNA (Figure 7b), suggesting that NF-κB activation is required for NALP3 inflammasome activation.